The invention provides a device and process for decellularizing, and/or devitalizing, tissue grafts; and/or recellularizing, essentially acellular or devitalized tissue grafts, including for example essentially acellular or devitalized vascular tissue grafts, where the graft is derived from a human or animal sources, or is as constructed using any number of tissue engineering methodologies.
Vascular grafts include a wide variety of natural and synthetic tubular structures that may or may not contain valves. Valves in these tubular structures are usually intended to direct the flow of blood (or other nutrient materials) in one direction by preventing the backward flow of this (these) liquid solution(s). Examples of valved tubular structures include aortic, pulmonary, and mitral valves present in the hearts of most vertebrate animals and veins used to return blood flow from the periphery of the body to the heart for recirculation.
Vascular grafts constructed of synthetic materials include devices constructed from man-made polymers, notably Dacron and Teflon in both knitted and woven configurations such as those marketed by W.L. Gore, Inc. and Impra, Inc. where various forms of polytetrafluoroethylene (PTFE) are molded into a wide array of tubule structures (see for example U.S. Pat. Nos. 4,313,231; 4,927,676; and 4,655,769.
Natural vascular grafts, taken in the context of the invention, include valved and non-valved tubular structures obtained by methodologies broadly classified under the term xe2x80x9ctissue engineeringxe2x80x9d. Notably, tissue engineered blood vessels such as described in U.S. Pat. Nos. 4,539,716, 4,546,500, 4,835,102, and blood vessels derived from animal or human donors such as described in U.S. Pat. Nos. 4,776,853, 5,558,875, 5,855,617, 5,843,181, 5,843,180, and a pending patent application entitled xe2x80x9cA Process for Decellularizing Soft-Tissue Engineered Medical Implantsxe2x80x9d (patent application Ser. No. 09/528,371 incorporated herein in its entirety). The present invention involves vascular grafts derived using a novel process associated with tissue engineering as well as a novel bioreactor device for use in the process.
Tissue engineered natural vascular grafts, hereinafter vascular grafts, can be manufactured by processing of natural vascular grafts (including for example, veins, arteries, and heart valves.) with the objective of removing the cellular elements without damaging the matrix structure of that tissue-a xe2x80x9creductionistxe2x80x9d approach. This approach is generally referred to as decellularization and is the subject of several patents, of which U.S. Pat. No. 4,801,299 by Brendel and Duhamel is considered as one of the earliest such patents, and pending patent applications as described above. Decellularization of tissues has been attempted by incubating tissues in the presence of detergents, both anionic and nonionic, with and without digestion of nucleic acids.
Tissue engineered natural vascular grafts have also been constructed using a xe2x80x9cconstructionistxe2x80x9d approach. This approach involves the extraction of natural cellular and matrix components to obtain purified (or partially purified) fractions and then using these fractions to reconstruct a vascular graft from individual components. Alternatively, specific components of a vascular graft, for example collagen(s), can be obtained using recombinant DNA technologies and such highly purified and homogeneous materials used in the construction of natural vascular grafts via tissue engineering. Methods and materials for 3-dimensional cultures of mammalian cells are known in the art. See, U.S. Pat. No. 5,266,480. Typically, a scaffold is used in a bioreactor growth chamber to support a 3-dimensional culture. The scaffold can be made of any porous, tissue culture compatible material(s) into which cultured mammalian cells can enter and attach.
Both the reductionist and constructionist approaches are attempts to provide an acellular matrix that can be used directly as an acellular graft.
The invention provides a bioreactor approach to reseeding of vascular grafts, such as a decellularized aortic heart valve. The approach involves removal of the basement membrane by enzymatic digestion. This removal of basement membrane is followed by pressure-differential induced movement of fibroblastic cells in a solution into the matrix structure and reendothelialization by incorporation of endothelial cells into a collagenous/noncollagenous solution. This latter solution is compacted, as necessary, by xe2x80x9cpressurexe2x80x9d binding of this mixture onto the luminal surface to recreate a xe2x80x9cbasement membranexe2x80x9d containing endothelial cells. Cells are induced to resume metabolic activities following treatment with specific growth factors, for example fibroblast growth factor, or platelet aggregation under a pulsatile flow of nutrient solutions. The novel design of the bioreactor facilitates the processes described in the present invention.
According to one aspect of the present invention, a device and process is described for recellularizing and reendothelializing essentially acellular or devitalized tissue grafts including vascular grafts for use in replacement of defective tissues including for example defective heart valves and vascular conduits. The device is a bioreactor designed to facilitate selected steps in the processing such as recellularization and reendothelialization. The process includes several steps which may be conducted outside of the bioreactor and several steps which may be conducted inside of the bioreactor such that most of the invention is carried out in a closed processing system that will dramatically restrict contamination by microbiological and chemical/biological elements. The process comprises the following steps:
1) use of an essentially acellular vascular graft, such as a heart valve, whether constructed as an acellular graft using tissue engineering methods or decellularizing a native vascular graft using methods known in the art;
2) attaching the acellular graft into the bioreactor by attaching the graft directly to the inlet and outlet port connections or by sewing/attaching the graft to sewing rings and attaching the sewing rings to the inlet and outlet port connections and closing the unit as illustrated in the attached figures;
3) optionally treating the acellular graft with various growth and/or differentiation factors, such as fibroblast growth factor (FGF), polylysine, hyaluronins, proteoglycans, RGD-containing peptides, sodium dodecylsulfate, or suramin, to achieve binding in the tissue matrix;
4) washing the acellular graft with an appropriate aqueous solution to remove unbound, or loosely bound growth and/or differentiation factors;
5) debriding the basement membrane using proteolytic enzymes, for example dispase and/or collagenase, to achieve total or partial removal of the basement membrane lining the luminal surface of the acellular graft;
6) washing the acellular graft with an appropriate aqueous solution to remove excess proteolytic enzymes;
7) seeding the acellular graft with a fibroblastic cell population, allogenously or autogenously derived, using a positive pressure mediated infusion of cells into the tissue matrix spaces;
8) washing the recellularized graft with an appropriate iso-osmotic solution such that only the luminal volume and the volume outside of the vascular graft are replaced and no additional pressure flow occurs across the matrix of the recellularized graft;
9) seeding the recellularized graft with an endothelial cell population, allogenously or autogenously derived, using a viscous collagenous/noncollagenous mixture containing the endothelial cells;
10) partially pressurizing the luminal volume to compress the viscous collagenous/noncollagenous/endothelial cell mixture onto the luminal surface of the now recellularized and reendothelialized graft;
11) washing the now recellularized and reendothelialized graft to remove excess viscous collagenous mixture;
12) applying a slow pulsatile flow of nutrient rich and growth factor containing medium, optionally containing allogenous or autogenous platelets, to establish a functionally viable cell population in the graft such that the fibroblastic cell population and the endothelial cell population establish a viable vascular graft;
13) applying a pulsatile flow of nutrient rich medium at a flow rate appropriate to provide a stress field in the tissue graft appropriate to the physiological stimulation of the cell population in that tissue graft;
14) Preserving the now recellularized and reendothelialized graft by methods know in the art of cryopreservation, cold-storage preservation, and/or nutrient-culture preservation, or directly shipping and implanting the graft post recellularizing/reendothializing.